The growth and development conditions in mouse offspring derived from ovarian tissue cryopreservation and orthotopic transplantation

Abstract

Purpose

To investigate the potential development or metabolic risk in offspring derived from mice with transplanted frozen-thawed ovarian tissue.

Methods

Mice ovaries were intervened by vitrification (group V) and slow-freezing (group S) cryopreservation and orthotopic transplantation. Orthotopic transplantation of fresh ovarian (group F) and natural mating (group C) served as control groups. The fertility restoration and health conditions of generations were assessed by offspring counts, anti-fatigue and motor ability, and organ morphology. The methylation rate and expression level of imprinted genes (IGF2R, H19, SNRPN, and PLAGL1) were used to predict the potential risk of development in transplanted generations.

Results

Both the percentage of normal morphological follicles in different developmental periods and the litter size of receipt mice were comparable in all three transplanted groups. There was no significant difference in offspring mice’s birth defects, body weight gain, anti-fatigue ability, or exercise capacity among the four groups. The methylation rate of IGF2R, H19, and PLAGL1 showed a significant variation in cryopreservation groups as compared with control groups, as well as a difference in gene expression. The SNRPN appeared to be stable in methylation status. There were no differences in mRNA expression in all groups.

Conclusions

The different ovarian tissue cryopreservation methods did not influence either maternal fertility function or offspring growth. However, these technologies could affect the methylation rate and expression level of some development-related imprinting genes in the offspring, which may lead to some indeterminacy risk.

Electronic supplementary material

The online version of this article (10.1007/s10815-020-01734-5) contains supplementary material, which is available to authorized users.

Introduction

The advances in cancer treatment have led to an increased survival rate in young patients. However, radiotherapy or chemotherapeutic agents may cause endocrine dysfunction and premature ovarian failure in not only child-bearing age but also prepubertal female [1, 2]. These adverse effects will affect the quality of life and subsequently induce infertility of female patients.

Cryopreservation and transplantation of ovarian tissue have been proved a valid method for the rehabilitation and fertility preservation of patients with ovarian failure. Following Chen et al. [3] who employed a vitrification method named direct covering vitrification (DCV), we have also explored a vitrification method named needle immersed vitrification (NIV), which has achieved initial success in reducing the concentration of cryoprotectant, preserving ovarian tissue, and restoring ovarian function post-transplantation. The procedure of this method has been detailed described in our previous studies [4–7]. However, either traditional slow-freezing or the vitrification method could lead to cryoinjury and induce potential adverse events from numerous unphysiological operations and effects of physicochemical factors (such as in vitro culture, cytotoxicity reagents, and sudden change in either temperature or osmotic pressure) during the process of ovarian tissue cryopreservation and recovering. Nowadays, according to the recommendation from the American Society of Reproductive Medicine (ASRM) and the American Society of Clinical Oncology (ASCO), the ovarian tissue cryopreservation and transplantation has been endorsed to preserve fertility function for female patients with cancer [8, 9]. However, the lower usage rate of ovarian tissue cryopreservation (3.9~4.6%) and live-birth rate after transplantation (nearly 31%) lead to limited literatures on the long-term fertility outcome of transplanted receipts or the healthy conditions of their offspring [10]. The safety issue of offspring derived from ovarian tissue cryopreservation and transplantation is still ambiguous.

In this study, we evaluated the fertility restoration in ovarian transplantation recipient and the health conditions of filial generations, including anti-fatigue and motor ability exercise, and morphological assessment of different organs. We further investigated the conditions of four development-related imprinted genes IGF2R (insulin-like growth factor 2 receptor), H19 (H19 imprinted maternally expressed transcript), PLAGL1 (pleomorphic adenoma gene-like 1), and SNRPN (small nuclear ribonucleoprotein polypeptide N) in several vital organs, which involved in fetal growth promotion, postnatal growth, or energy homeostasis, trying to provide some in vivo data for evaluating the lifelong safety of ovarian tissue cryopreservation and transplantation (OCT).

Materials and methods

Animals

Four-week-old C57BL/6J mice (black coat hair, BC) and the albino mutation strain C57BL/6J-Tyrc-2J-J (white coat hair, WC) were purchased from The Jackson Laboratory, USA, and bred in SPF Institutional Animal Care and Use Center, West China Second Hospital, Sichuan University. All mice were fed with chow diet and water ad libitum and kept in cages with controlled temperature (18–22 °C), humidity (60–80%), and light (12 h light, 12 h dark). This study has been approved by the Institutional Animal Care and Use Committee of West China Medical Center, Sichuan University.

Experimental design

As the ovary donor, 4-week-old BC female mice were evenly divided into 4 groups randomly. Group 1 was set up as control group, which used 8-week-old female mice mating with WC male mice directly (group C). The other three groups were performed with different methods of ovarian tissue cryopreservation and transplantation separately, including slow-freezing (group S), needle immersed vitrification (group V), and fresh ovarian transplantation (group F). The frozen-thawed ovaries and fresh ovaries were orthotropic transplanted into the ovarian bursa of 8 weeks WC female mice respectively. Recipient mice mated with 8-week-old WC male mice 2 weeks after transplantation, and the black hair offspring from the mice with transplanted ovaries were used as study subjects (Fig. 1a–d).

Fig. 1.

Ovarian grafts to recipients mouse and the birth defect in offspring. a, b, c The surgery of ovarian transplantation to WC receipt mouse. d Litter condition of receipt mice. e Rectum valgus deformity in one black hair offspring mouse

Ovarian tissue slow-freezing and thawing

Two to three intact C57BL/6J mouse ovaries were placed in a 1.8 ml cryovial (Nunc, Roskilde, Denmark) containing with 1 ml L-15 medium (Gibco, Grand Island, NY, USA) supplemented with 0.1 M sucrose (Sigma-Aldrich, St Louis, MO, USA), 10% FBS (Gibco), and 1.5 M DMSO (Sigma-Aldrich). After incubating with cryoprotectant solution at 4 °C for 30 min, the cryovial was placed in a programmable freezer (Biomed Freezer Kryo 10, Series II, Planer, UK). The slow-freezing procedure was as follows: cooled at a rate of − 2 °C/min from 4 °C to − 7 °C and held for 5 min → seeded manually and maintained at − 7 °C for another 10 min → cooled at a rate of − 0.3 °C/min to − 40 °C → cooled at a rate of − 10 °C/min to − 140 °C. Finally, the samples were stored in liquid nitrogen for at least 1 week.

For thawing, the vials were taken out from liquid nitrogen and held in air for 20 s, then transferred to a water bath for 20~30 s at 37 °C. After that, the ovaries were taken out from vials and incubated with L-15 medium supplemented with 0.1 M sucrose, 10% FBS, and 1.0 M DMSO for 5 min, and washed by a stepwise manner (0.1 M sucrose + 1.0 M DMSO, 0.1 M sucrose + 0.5 M DMSO, 0.1 M sucrose) for 5 min each [5].

Ovarian tissue NIV cryopreservation and thawing

Three to four intact 4-week-old C57BL/6J mouse ovaries were held in a row by an acupuncture needle (0.18 mm in diameter and 1.3 mm in length, HuaTuo, Medical Device Co. Ltd., Suzhou, China) in L-15 medium supplemented with 10% FBS. Then, the above ovaries were dehydrated with a two-step approach: (1) an equilibration solution containing 20% FBS, 7.5% (v/v) DMSO, and 7.5% (v/v) EG (Sigma-Aldrich) in DPBS for 10 min; (2) a vitrification solution containing 0.5 M sucrose, 15% DMSO, and 15% EG for 2 min at room temperature. The remaining solution on the needle was removed softly by aseptic absorbent gauze, and the needle was plunged into liquid nitrogen directly.

For thawing, the ovaries held by a needle were taken out from liquid nitrogen and immersed into 1 M sucrose at 37 °C for 5 min and 0.5 M and 0.25 M sucrose at room temperature for 5 min each successively, then washed with DPBS supplemented with 20% FBS three times, and finally incubated at 37 °C with 5% CO₂ incubator for at least 20 min before transplantation [5].

Transfer to the ovarian bursa

Fresh and freeze-thawed ovaries from donors were transplanted into the ovarian bursa of C57BL/6J-Tyrc-2J-J mice following the bilateral grafting procedure by Liu et al. [11] with some modification. Briefly, the recipients were anesthetized by i.p. injection of 1% pentobarbital. The ovarian bursa was accessed through a dorsally horizontal incision, which was extended below the fat pad, and the bursal membrane was reflected over the ovary. A pair of fine watchmaker’s forceps was used to grip the hilum, and a second pair of forceps was used to sever the native ovary from the hilum. Pressure was applied for 10 s to control bleeding. The donor ovary was placed within the bursal cavity, with the bursal membrane replaced and closed with 9–0 nylon sutures before returning it to the peritoneal cavity. Both ovaries of the recipient animal were removed partly and replaced with donor ovaries.

Evaluation of receipt mice fertility restoration and offspring mice health condition

We conducted comparative analysis of ovarian follicle morphology, litter size, birth defect, growth, development, and body weight of black hair offspring mice. Movement coordination was evaluated with a rotating stick test. Anti-fatigue ability was evaluated with load swimming test.

Recipient mice ovaries (2 mice per groups) were removed and fixed in 10% neutral-buffered formalin solution for 24 h at 4 °C, dehydrated, paraffin-embedded, and cut into 5-μm-thickness sections, then the sections were stained with hematoxylin and eosin (H&E). Every fifth section of each ovary was analyzed for follicle counting. To avoid counting follicles more than once, only follicles with a visible nucleus were counted [5].

The primordial follicles were defined as those containing one layer of flattened, or a mixture of flattened and cuboidal, pre-granulosa cells surrounding the oocytes; the primary follicles as those with one-layer cuboidal follicular cells; the secondary follicles as those where the oocytes were surrounded by two or more layers of cuboidal granulosa cells without an antrum; antral follicles are those with multiple layers of cuboidal granulosa cells with an antrum [12].

Organs from offspring mice including the brain, heart, liver and kidney were also fixed in 10% neutral-buffered formalin solution then further sections for H&E staining and morphology assessment.

Examination of filial mice’s gene methylation and expression

The methylation and expression status of imprinted genes (IGF2R, H19, SNRPN, and PLAGL1) were detected and compared in both ovary-transplanted mice and control mice. DNA was isolate from tissue specimen with DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). Bisulfite treatment for methylation analysis followed the manufacturer’s protocol of MethylDetector™ Bisulfite Modification Kit (Takara, Dalian).

Total RNA was isolated from tissues with RNAiso Kit (Takara) and converted to cDNA using PrimeScript RT reagent Kit DRR037 (Takara). A real-time qPCR was performed with ABI Prism 7500 System (Applied Biosystems Inc., USA) and SYBR® Green Realtime PCR Master Mix (Takara), under the following parameters: 95 °C for 10 min for denaturation, 40 cycles of 95 °C for 10 s, and 60 °C for 34 s. Supplemental Table 1 shows the detail of primer sets used for each gene.

Statistical analysis

Quantitative data were expressed as mean ± SD. One-way analysis of variances (ANOVA) was conducted to assess differences between the groups. Count data are expressed as row x list. A chi-squared test was used to compare the rate of each group. P < 0.05 was considered as statistically significant. The data were analyzed by SPSS 13.0 software.

Results

The fertility restoration of frozen-thawed ovarian transplantation recipient mice

After transplantation, the body weight of the recipient mice decreased in short term, but the change diminished after 2 weeks compared with the control mice. The histological structure of ovarian tissue was roughly normal after thawing and incubation. We also compared the percentage of normal follicle morphology in different developmental periods among three transplantation ovaries (fresh control and frozen-thawed ovarian orthotropic transplantation groups). There was no significant difference in the percentage of normal follicle morphology among grafting tissues. At all stages, follicles in cryopreserved ovaries were preserved with satisfaction compared with fresh control (Table 1). After mating, the litter sizes (total/black hair) of each group were 79/37 in fresh, 64/30 in vitrification, and 68/32 in slow-freezing respectively. We observed no significant difference either in counts of total offspring or in offspring with transplanted ovaries (Table 2).

Table 1.

The number and percentage of normal follicle morphology in different developmental periods among three transplanted groups

Groups	Primordial follicles		Primary follicles		Secondary follicles		Antral follicles
	Number of normal follicles	Percentage of normal follicles	Number of normal follicles	Percentage of normal follicles	Number of normal follicles	Percentage of normal follicles	Number of normal follicles	Percentage of normal follicles
Group F	113.17 ± 14.85	91.7 ± 3.67%	55.67 ± 2.12	83.7 ± 4.89%	33.5 ± 9.19	77.7 ± 5.03%	14.33 ± 2.83	71.5 ± 8.08%
Group V	99.5 ± 30.41	86.1 ± 5.03%	42 ± 12.73	82.9 ± 4.25%	21.67 ± 3.54	75.8 ± 4.64%	13.5 ± 5.66	69.8 ± 9.02%
Group S	111.5 ± 47.38	83.9 ± 6.64%	61 ± 15.56	82.0 ± 2.58%	24.17 ± 22.63	74.1 ± 4.71%	9.83 ± 0.71	67.3 ± 12.08%
P		0.056		0.737		0.453		0.099

Number and percentage data were expressed as mean ± SD

Table 2.

The counts of total offspring and transplanted ovaries offspring in three transplanted groups

Groups	Total litter size		P	Survived BCO (number)	P
	BCO	WCO
Group F	37	42	*0.995	33	0.245 #0.456
Group V	30	34		22
Group S	32	36		26

BCO offspring mice with black coat hair, WCO offspring mice with white coat hair

*P value for the BCO counts comparison in three groups

^#P value for group V versus group S

Fecundity, growth, and development of offspring mice

The numbers of black hair offspring mice survived to adulthood were 33 in fresh, 22 in vitrification, and 26 in slow-freezing. Although the survival rates of offspring from ovary-transplanted groups showed a declining trend in group V (73% in vitrification, 89% in fresh, 81% in slow-freezing), no significant difference was present after statistical analysis (Table 2). All mice showed no apparent birth defect except one from group V had the rectum valgus deformity (Fig. 1e). No significant difference in weight gain was observed in four groups (P > 0.05, Fig. 2a, b). Meanwhile, there was no significant difference in anti-fatigue ability (load swimming test) or motor ability (rotating stick test) in four groups (P > 0.05, Fig. 2c, d).

Fig. 2.

The healthy conditions offspring mice in four experimental groups. Body weight gain of offspring female (a) and male (b) mice. Loading swimming test (c) and rotating stick test (d) in adults

In addition, the morphological presentation of some vital organs (the brain, heart, liver, and kidney) in the transplanted groups showed similarity to the control group (Fig. 3). Similarities was also observed in organ and body weight ratio.

Fig. 3.

Histological examination of different organs from transplantation offspring mice and control offspring mice. From left to right were groups V, S, F, and C respectively

Changed methylation status in imprinted gene of mice

The IGF2R gene methylation of liver tissue in the cryopreservation and transplantation group was significantly declined compared with group C (P < 0.001). No significant difference (P = 0.069) was observed between groups V and S, although the methylation rate was slightly lower in group V. However, no similar phenomenon was observed in brain tissue. The IGF2R gene methylation rates showed no significant difference among the four groups (P = 0.058) (Table 3, section A).

Table 3.

The methylation status (A) and mRNA expression level (B) of four imprinted genes in liver and brain tissues

	Tissue	Group	IGF2R	P	H19	P	SNRPN	P	PLAGL1	P
A	Liver	Group C	64.33% ± 6.26%	< 0.001 *0.069	$#44.19 ± 5.14%	0.040 *0.071 #0.031 $0.624 &0.110	68.41% ± 22.29%	0.953 *0.761	@$#70.25% ± 7.56%	0.001 $0.010 *0.295 #0.868 @0.021
		Group V	*33.87 ± 4.37%		#*60.04 ± 6.64%		*72.53% ± 13.26%		$*48.98% ± 2.87%
		Group S	*43.94 ± 5.54%		$*@46.76% ± 6.63%		*76.19% ± 14.34%		@*52.29% ± 3.80%
		Group F	56.78% ± 3.83%		@59.38% ± 8.38%		78.90% ± 6.78%		#69.09% ± 8.51%
	Brain	Group C	62.97% ± 3.65%	0.058 *0.819	61.75% ± 2.94%	0.346 *0.223	74.25% ± 4.03%	0.983 *0.808	@$#80.64% ± 7.77%	< 0.001 $< 0.001 *0.032 #0.805 @< 0.001
		Group V	*48.5 ± 4.40%		*59.59% ± 10.50%		*73.80% ± 2.29%		$*57.53% ± 3.66%
		Group S	*49.63 ± 5.96%		*50.80% ± 2.03%		*74.67% ± 5.34%		@*46.62% ± 4.56%
		Group F	54.81% ± 8.23%		61.28 ± 11.14%		73.34% ± 5.08%		#78.99% ± 7.63%
B	Liver	Group C	1 ± 0.13	< 0.001 *0.013	1 ± 0.39	< 0.001 *1.000 #0.177	1 ± 0.01	0.202	1 ± 0.08	0.013 *0.047
		Group V	*1.5 ± 0.04		#*0.22 ± 0.01		1 ± 0.02		*1.55 ± 0.20
		Group S	*1.36 ± 0.04		#*0.22 ± 0.03		1.01 ± 0.02		*1.98 ± 0.12
		Group F	1.11 ± 0.07		#0.16 ± 0.06		1.03 ± 0.02		1.13 ± 0.28
	Brain	Group C	1 ± 0.07	0.795	@#1 ± 0.07	< 0.001 *< 0.001 #0.097 @0.043	1 ± 0.02	0.809	1 ± 0.21	0.002 *0.047
		Group V	1.16 ± 0.21		#*1.24 ± 0.07		1.06 ± 0.06		*1.46 ± 0.26
		Group S	1.19 ± 0.16		@*0.68 ± 0.05		1.05 ± 0.08		*1.91 ± 0.09
		Group F	1.07 ± 0.11		1.43 ± 0.03		1.06 ± 0.01		1.02 ± 0.05

The methylation rates of H19 gene in liver tissues of filial generation mice were different among the four groups. However, statistical analysis showed only group V with a significant increase in comparison with group C (P = 0.031), while in brain tissue, there was no significant difference among each group (P = 0.346) for H19 gene (Table 3, section A).

The SNRPN gene methylation rate in liver and brain tissue suggested favorable consistency in each group. There was no significant difference among each group (P = 0.953, 0.761) or between the cryopreservation and transplantation groups (P = 0.808) (Table 3, section A).

The PLAGL1 gene methylation rate in liver tissue was significant different among each group (P = 0.001), and similar results were found in brain tissue (P < 0.001). When making pairwise comparison among the four groups, we found that both two cryopreservation groups were significantly declined comparing with control group (P = 0.021 for group S, P = 0.01 for group V), and no statistical difference was shown in either two cryopreservation groups or group F comparing with group C. We further compared the intervention groups with the control group separately and found that the PLAGL1 gene methylation rate in brain tissue in two ovarian tissue cryopreservation transplantation groups was significantly lower than that in group C (P < 0.001), which was more obviously lower in group S (Table 3, section A).

Detection of imprinted gene expression

RNA was extracted from different tissues (liver and brain) in each group to examine the expression levels of imprinted genes. In the liver, the expressions of both IGF2R and PLAGL1 in the ovarian tissue cryopreservation groups (groups S and V) were significantly higher than in group C or group F; however, IGF2R was more strongly elevated in group V unlike PLAGL1 which was elevated in group S more; for H19 gene, the expressions in the ovary-transplanted groups were significantly decreased compared with that in the control group (P < 0.001), with neither significant difference among three transplantation groups (P = 0.177), nor significant difference between group S and group V (P = 1.000); no significant difference was either found in the expression of SNRPN among all groups (P = 0.202) (Table 3, section B).

In brain, both IGF2R (P = 0.795) and SNRPN (P = 0.809) gene expressions suggested no statistic difference among each group; the H19 gene expression significantly decreased in group S compared with either group C (P = 0.043) or group V (P < 0.001); compared with group C and group F, the expressions of PLAGL1 in frozen ovary–transplanted groups were increased significantly, especially in group V (Table 3, section B).

Relationship between methylation status and mRNA expression level in imprinted genes

The IGR2R (Fig. 4a–d), SNRPN (Fig. 4i–l), and PLAGL1 (Fig. 4m–p) gene expression and methylation of liver and brain tissue from filial generation mice showed satisfied consistency. The methylation rate of IGF2R gene in liver tissue (Fig. 4a) of the cryopreservation and transplantation group significantly declined in comparison with group C; meanwhile, the expression of IGF2R gene mRNA (Fig. 4b) was elevated significantly. In brain tissue, the IGF2R gene methylation (Fig. 4c) showed no significant difference, so was the mRNA expression (Fig. 4d). Nevertheless, the H19 gene showed inconsistent results between the methylation rate and gene expression level either in liver or in brain tissue of filial generation mice (Fig. 4e–h). These results suggested the unknown and numerous influence factors of H19 expression, which requires further investigation.

Fig. 4.

The methylation rate and mRNA expression level of four imprinted genes. To compare the methylation status of imprinted genes among four groups: IGF2R in the liver (a) and brain (c); H19 in the liver (e) and brain (g); SNRPN in the liver (i) and brain (k); PLAGL1 in the liver (m) and brain (o). The comparison of imprinted gene mRNA expression among four groups: IGF2R in the liver (b) and brain (d); H19 in the liver (f) and brain (h); SNRPN in the liver (j) and brain (l); PLAGL1 in the liver (n) and brain (p); *Compared with control group (group C), P < 0.05; ^#Compared with group F, P < 0.05

Discussion

Because of the lower usage rate of orthotopic transplantation after ovarian tissue cryopreservation (1.9~4%) and rare ultimately generated pregnancy and delivery rates of participants (31~37%) [13–18], few literatures reported the fertility outcome of maternal or health conditions about transplanted offspring [19], especially the comparison between slow-freezing and vitrification. In this study, we made great effort to build up an optimal frozen-thawed ovarian tissue grafting animal model, in order to have sufficient animals to perform the subsequent experiment. After several trials, we observed that keeping a little amount of endogenous ovarian tissue in situ of recipients and transplanted into the ovary could have the effects of facilitating graft survival, making target organ stimulate continuously by ovarian hormone, and promoting the ovary functional recovery after surgery as soon as possible.

There are two cryopreservation methods employed in fertility preservation: slow-freezing and vitrification [20]. After decades of clinical practice, vitrification showed a superior clinical outcome (higher survival rate and pregnancy rate, etc.) in preserving oocyte [21–23] or embryo [24, 25]. As the only available option of preserve fertility for prepubertal females [26], some studies also compared the clinical outcomes between the above two methods in ovarian tissue cryopreservation in recent years, attempting to find a better choice. However, to date, no evidence of vitrification is superior to traditional slow-freezing [27, 28]. In order to provide more experimental data, we also made a comparison in both fertility restoration and offspring outcome of these two cryopreservation methods in this study.

During the long-term follow-up of different intervention groups, we found the opposite outcomes of fertility function in each time course between group V and group S. In short-term ovarian rehabilitation, group V was superior to group S. This result might be related to little ice crystal formation by ultra-rapid cooling technique during vitrification procedure, which could preserve extracellular matrix of ovarian tissue at maximum and benefit from the formation of local microcirculation in grafts. This result is consistent with our previous publications [4]. However, as time went on, we observed that group S seemed to be better in the long-term preservation of reproductive function than group V. Group S mice were able to get pregnant and delivery transplanted offspring after 12 months, while group V stopped to be pregnant and childbirth in the observation to 8–10 months after operation. This contrary result might be due to the higher concentration of cryoprotectant for vitrification (such as higher concentration of DMSO and EG used in the dehydration step), which could increase the cytotoxicity than the slow-freezing method. Therefore, in order to decrease the dose of the cryoprotectant to reduce the toxicity and maintain the long-term reproductive function, the ovarian tissue cryopreservation technology should be further optimized.

To date, neither basic research nor clinical practice has reported any information about the effect of ovarian tissue cryopreservation on offspring’s malformation. In our study, only one case of vitrification offspring suffered with rectal valgus deformity after birth. All the other filial generation performed no obvious birth defect either in control or in intervention groups. No significant difference was observed in the death of newborn mice, stillbirth, infanticide, and the rate of survival to adulthood among each group. Previous studies have shown a higher risk of metabolic dysfunction for offspring after accepting the traditional assisted reproductive technology (like in vitro fertilization and embryo transplantation), such as abnormal weight (underweight or obesity) and increasing risk of nervous/sports system disorders [29, 30]. In this study, we also observed the weight change of filial generation and evaluated the movement coordination and anti-fatigue ability of offspring mice. From the results, we found no significant difference in birth weight or weight change among each group. In rotating rod test and weight loading swimming experiment, the offspring from group S and V demonstrated the familiar sport function with the control group. These phenomena were different from previous reports on ART offspring. Whether ovarian freezing and thawing transplantation technology has more advantages in the safety of progeny growth and development required further studied to verify.

DNA methylation, which is crucial for regulating the epigenetic alterations of gene in mammals, could be reprogrammed in two developmental periods (germ cells and preimplantation embryos) [31]. The traditional ART (in vitro fertilization and embryo transplantation) will affect those two important periods and lead to some severe birth defect in offspring, such as large offspring syndrome, metabolic disorder, Beckwith-Wiedemann syndrome, or tumorigenesis [32–35]. Since ovarian tissue cryopreservation could avoid the above two periods, we suspected that this technology might be safer in epigenetic states comparing with IVF and embryo transplantation. In order to verify this hypothesis, we chose some development-related imprinted genes including IGF2R, H19, SNRPN, and PLGAL1, and made a comparison of methylation status and gene expression among four groups. We found that in some vital organs (the brain and liver), the IGF2R, H19, and PLAGL1 showed varying degree changes in both the methylation rate and mRNA expression in ovarian tissue cryopreservation groups, which may indicate a growth-related modification or tumorigenesis tendency in offspring [36]. SNRP imprinting plays an important role in cognitive ability and animal behavior [37]. In our study, both methylation rate and mRNA expression of SNRPN gene were stable, unaffected by the schedule of operation. This result was consistently with previous publications, which demonstrated that paternal only reprogramming was the key point of SNRPN-associated diseases [38]. Based on the above results, although no clinical data showed a birth defect of ovarian tissue cryopreservation and transplantation offspring, it seems that the safety of this technology is still controversial. More studies are required to explore this topic in the future.

Conclusions

The different ovarian tissue cryopreservation methods did not influence either maternal fertility function or the growth of offspring. However, these technologies could affect the methylation rate and expression level of some development-related imprinting genes in the offspring, which may lead to some indeterminacy risk.

Funding information

This study was supported by the National Natural Science Foundation of China (31201117, 81673032) and Key Research and Development Program of Science and Technology Department, Sichuan Province (2019YFS0418).

Compliance with ethical standards

This study has been approved by the Institutional Animal Care and Use Committee of West China Medical Center, Sichuan University.