Perinatal Outcomes of Pregnancies After Infertility Treatment Using Assisted Reproductive Technologies With Cryopreserved Embryo Transfer

The paper presents an analysis of the data on obstetric and perinatal outcomes of pregnancies after infertility treatment by the methods of assisted reproductive technologies (ART) using the transfer of one selected cryopreserved embryo, particularly the outcomes of spontaneous and induced singleton pregnancies. The clinical and anamnestic data of patients were analyzed. The course of pregnancy, childbirth and the state of the newborn were assessed using the data of individual medical record of the pregnant woman and discharge report from the maternity hospital. The criteria for assessing obstetric and perinatal outcomes included: the frequency of normal physiological and preterm delivery, the method of delivery and the weight of children at birth. Factors infl uencing the course of pregnancy and the condition of children born after natural conception and the use of ART have been determined. It was found that in a group of patients with transfer of a cryopreserved embryo of delivery by cesarean section are performed more often. The body mass index of newborns was statistically and signifi cantly higher than in the groups with spontaneous and induced pregnancies: (3,653.3 ± 550.5), (3,420.2 ± 547.1) and (3,087.1 ± 704.7) g, respectively.


Материалы и методы
В работе проведен ретроспективный анализ акушерских и перинатальных данных медицинских карт при информированном согласии пациенток, проходивших курс лечения бесплодия методами ВРТ в 2015-2016 гг.в клинике репродуктивной медицины «Надия» (г.Киев, Украина).Все манипуляции с гаметами и эмбрионами проводили согласно положений «Конвенции Совета Европы о защите прав и достоинства человека в связи с применением достижений биологии и медицины» (Овьедо, 1997), доклада Главного комитета pre-implantation genetic diagnosis of embryos and thereby increased the effi ciency and safety of ART [13,14].The strategy of transferring a single embryo to avoid multiple pregnancy, which is a complication of ART, predetermines the need to preserve the remaining embryos [4].In case of the absence of pregnancy after course of treatment or the need for a delayed embryo transfer in the next favorable ovulatory cycle, the frozen-thawed embryos is transferred.The number of therapeutic protocols with the transfer of cryopreserved embryos enhanced from 2,275 up to 5,868, i. e., 2.57 times in Ukraine [17].It has been shown that the frequency of pregnancy onset after transfer to the uterine cavity of cryopreserved embryos in the natural cycle is higher than in the stimulated embryo [10].This is facilitated by the improvement of cryopreservation techniques, rehabilitation of cells after freezing-warming and optimization of the conditions for cultivation of cryopreserved embryos in vitro [5,16].Taking into account the age, somatic anamnesis of women, the duration of infertile marriage, as well as the infl uence of suboptimal conditions of cultivation and aggressive factors of cryopreservation on preimplantation embryos, the study of the features of the course of pregnancies and childbirth outcomes gains an important scientifi c and clinical signifi cance.
The world scientifi c literature presents the data on obstetric and perinatal outcomes of spontaneous pregnancies and after ART, however, no such an analysis was conducted in Ukraine [1,8,11].
In connection with the foregoing, the goal of the study was to compare the obstetric and perinatal outcomes of spontaneous or induced pregnancies, and those obtained after the transfer of one cryopreserved embryo of patients undergoing infertility treatment with ART methods.
Акушерские и перинатальные исходы оценивали по следующим критериям: частота нормальных физиологических родов (после 37 недель беременности) и преждевременные родораз-in the stimulated cycle in infertility treatment, the second group made 62 patients with a transfer of one cryopreserved embryo in the assisted cycle.To identify the risk factors and determine the features of the pregnancy course in the patients after ART, group 3 (n = 58) was composed, in which the singleton pregnancy oc-curred spontaneously.The analysis of clinical and anamnestic data included the average age of the mother at the time of childbirth, infertility duration, the body mass index (BMI) and extragenital pathology.Patients were excluded from the study according to the following criteria: age over 42 years, BMI more than 29 or less than 19 kg/m 2 , participation in the oocyte donation program.
All stages of ART were performed in accordance with standard protocols approved by the WHO and the Ministry of Health of Ukraine [3].The results of the presented work were obtained on the base of retrospective chart review.
Embryos were cryopreserved at the blastocyst stage, for this purpose they were placed into cryoprotective mixture of 7.5% ethylene glycol (EG) and 7.5% dimethylsulfoxide (DMSO) solutions for 10 minutes and then transferred to a cryoprotective mixture of 15% EG and 15% DMSO.The embryos were placed one by one on the surface of polypropylene open carrier Сryotec (Сryotec, Japan), which was rapidly immersed into liquid nitrogen [7].The samples were stored at a low-temperature bank from one month to fi ve years.The embryos were thawed rapidly, placing the carriers in to a 1 M sucrose solution pre-warmed up to 37°C.After a minute, the embryo was transferred to a medium containing 0.5 M sucrose solution, after 3 minutes it was moved to the culture medium (LifeGlobal, USA) in culture plates (Nunc, Denmark).Before the transfer to uterine cavity, the embryo was cultured for at least 3 h.
Patients with confi rmed clinical pregnancy were observed in the women prenatal department of the Clinic Nadiya.The course of pregnancy, childbirth and the condition of the newborn was assessed according to the data of the individual medical record of the pregnant woman and discharge report from the maternity hospital.
Obstetric and perinatal outcomes were assessed according to the following criteria: the frequency of normal physiological delivery (after 37 weeks of gestation) and premature delivery (up to 37 weeks gestation); method of delivery; body weight of children at birth, perinatal losses, birth traumatism.The clinical condition of children at birth was analyzed using the Apgar scores on the fi rst and fi fth minutes of life.An evaluation of the newborn functional  Таким образом, пациентки экспериментальных и контрольной групп не различались по возрасту, ИМТ, но у женщин групп 1 и 2 отмечен длительный срок бесплодного брака в отличие от группы пациенток, у которых беременность наступила спонтанно.
state was taken into account; as well as basic parameters of physical development; degree of maturity; somatic and neurological status; presence of malformations.
The gestational age (gestational age of the fetus) was the number of full weeks that passed from the fi rst day following the last menstruation and the birth, in case of spontaneous conception, or full weeks between the day of embryo transfer and the outcome of pregnancy plus 3 weeks in case of conception after ART.
The results were processed using software Stat-Soft Statistica (StatSoft, USA).Data group analysis was used to analyze descriptive statistics: the arithmetic mean (M), the standard error of the mean (SE), the standard deviation (SD), the distribution pattern.The parametric F-and t-test was used to assess the signifi cance of the diff erences in the data between the clinical groups, and p < 0.05 was considered as a criterion of statistical signifi cance.

Results and discussion
All women underwent general clinical and gynecological examinations.Patients of the studied groups did not diff er in age and BMI.In patients of groups 1 and 2, the duration of infertility was longer than in women of group 3.
Thus, the patients under study and the control groups did not diff er in age, BMI, but women of groups 1 and 2 had a long infertile marriage, in contrast to a group of patients whose pregnancy occurred spontaneously.
The survival rate of embryos after cryopreservation was 96%, and the frequency of pregnancy onset in patients of group 2 was 49.3%.
Pregnancy in Group 1 patients occurred against the background of diseases of the cardiovascular system in 48% of cases, in group 2 -51.4% and in group 3 -46.55%.A high incidence of diseases of the genitourinary system was noted in the natural conception group (29, 17 and 51.7% of pregnancies in groups 1-3, respectively).This is likely due to the fact that the patients before the cycle of programmed conception undergo mandatory clinical and laboratory examination.If there were the signs of infl ammation, infections of urinary tract, women undergoing ART had treatment with mandatory laboratory control, which was absent in the natural conception group.
respectively.In connection with ischemic-cervical insuffi ciency, a circular suture was applied in 3.2% of patients in group 1 and 5.7% in group 2. Because of shortening of the cervix, a pessary was applied to 9.7% of women in group 1; 5.7% of group 2 and 8.6% of group 3.The percentage of preterm delivery in groups 1 to 3 was 6.45, 14.2 and 8.6, respectively.
A high frequency of surgical delivery by cesarean section in groups 1 and 2 was noted.Indications for the surgery to women of these groups were fetal distress; abnomalities of labor activity according to partogram data, not amenable to drug correction; complicated delivery (clinically narrow pelvis, extensor insertions of the head).When comparing the groups with the use of ART, it was found that in the group with the cryopreserved embryo transfer, the frequency of surgical delivery was higher than after the embryo transfer in the stimulated cycle.
It should be noted that the diff erences in the evaluation of newborns according to the Apgar score were statistically insignifi cant (p > 0.05).
There are publications describing obstetric and perinatal outcomes in the ART cycles after the transfer of cryopreserved embryos with the use of slow cooling regimens and low concentrations of cryoprotectants, mainly 1,2-propanediol [12].In our study, all pregnancies in cryocycles were obtained after cryopreservation of the embryos by vitrifi cation, which involves the application of high concentrations of cryoprotectants.The fact of cytotoxic, mutagenic and teratogenic eff ect of high concentrations of DMSO is well known, therefore it was important for us to assess the state of health of children born after cryopreserved embryo transfer [4].
The question of the dependence of the body weight of the child at birth on the technologies used (in vitro culturing, cryopreservation) is discussed in many studies [2,1,11,12].For example, after the transfer of cryopreserved embryos, there was a decrease in the number of preterm births [12].F. Belva et al. [1] did not establish signifi cant diff erences in the body weight at birth of children obtained after cryo-and stimulated cycles.A higher body mass index of children born after the transfer of cryopreserved embryos was noted by S. Pelkonen et al. [11], which agrees with our data.The report of K. Takahashi et al. [15] underlined increased the incidence of preterm birth by 18.5% and low birth weight in children after using cryopreservation of embryos.In our study, there was no case of malformation of the fetus.This is perhaps due to the fact that the study group included patients with the transfer of one selected embryo.There are the reports of such perinatal consequences as an increased incidence of diseases and mortality, along with prematurity, immaturity and underweight.In our opinion, and in the opinion of other researchers, this is a consequence of the transfer of several embryos [6].The method of vitrifi cation does not increase the frequency of congenital malformations of children (1.18-1.42)%and does not exceed the parameters of the control group [9].
Thus, the study shows that cryopreservation factors do not increase perinatal risks and do not adversely aff ect the physical health of children.However, we believe that it is necessary to conduct a large multicenter population study on perinatal outcomes and development of children born after the application of ART technology in Ukraine.The data obtained will allow not only supplement the global statistics on the impact of cryopreservation factors on outcomes of childbirth and child development, but will also to determine the possibility of its application in Ukraine, since not only multifactor effect of cryopreservation should be taken into account, but also national and ecological features of the region.

Conclusions
In the group with the transfer of the cryopreserved embryo, delivery by cesarean section is performed more often, the birth weight of the newborn is signifi cantly higher than in the groups with spontaneous and induced pregnancies.Cryopreservation factors do not increase perinatal сы тела новорожденных статистически значимо выше, чем в группах со спонтанной и индуцированной беременностями.Факторы криоконсервирования не увеличивают перинатальные риски и не оказывают негативного влияния на физическое здоровье детей.risks and do not adversely aff ect the physical health of children.