Risk factors for twin pregnancy in women undergoing double cleavage embryo transfer

In vitro fertilization (IVF) has allowed millions of infertile couples to get conception since the year 1978 [1]. IVF is more commonly used in recent years due to the increase of infertile couples [2‐5]. However, IVF treatment is associated with the increased risk of several adverse obstetric and neonatal outcomes, including multiple pregnancy [6, 7], ovarian hyperstimulation syndrome (OHSS) [8], preterm birth [9, 10], low birthweight [11, 12], and small for gestational age [13‐15].

Two or more embryo transfers have been used to increase the success rate of live birth in traditional IVF strategy [16, 17]. However, the risk of multiple pregnancy is also increased following the transfer of multiple embryos [18]. Multiple pregnancy is associated with increased risks of premature birth, placental related diseases (preeclampsia, gestational hypertension, placental abruption), cesarean section, and postpartum bleeding [19‐24]. In addition, compared with singleton pregnancy, multiple pregnancy results in higher risks of several adverse neonatal outcomes, including low birth weight, fetal or neonatal death, and congenital malformations [25, 26]. Multiple pregnancy rate following IVF has been reduced over time due to the increased application of transfers with fewer and better-quality embryos [18, 27‐29]. More recently, elective single embryo transfer has been increasingly popular. However, the decision towards elective single embryo transfer or double embryo transfer remains inconclusive up to now [30, 31], which needs to be further explored.

Previous studies demonstrated that the factors associated with twin pregnancy in women undergoing IVF include the increased height of women (> 1.74 cm), the younger age of women, a higher quality of transferred embryos, a high number of retrieved oocytes (> 8) [32‐34]. However, due to the small sample size, the omission of some important covariables, and the heterogenous inclusion of participants, several potential risk factors for twin pregnancy may be neglected. Therefore, in this retrospective chort study, we aimed to explore the risk factors of twin pregnancy in women with double embryo transfer. We also investigated whether the risk factors of twin pregnancy were associated with the adverse outcomes, including preterm birth and low birth weight. We analyzed the data that include those with failed pregnancies after embryo transfer because we would like to highlight how these risk factors could be used as a tool for both pre-transfer and post-transfer counselling [35, 36]. This made our analyses more notable than previous studies which only looked at risk factors as a post-transfer counselling tool.

This retrospective cohort study was approved by the Ethics Committee of Women’s Hospital of Zhejiang University. A total of 15,800 women who underwent their first double embryo transfer (DET) cycle from January 2010 to December 2018 in Women’s Hospital, Zhejiang University School of Medicine were enrolled in this study. Women with the following cycles were excluded: preimplantation genetic test (PGT); donor cycles; cyeles transferred with one; cleavage embryo and one blastocyst; cycles transferred with double blastocyst. Women who resulted in pregnancy was excluded if they had a triple pregnancy, monozygotic twins, fetal reduction, or loss of follow up. Gonadotropin-releasing hormone (GnRH) agonist or GnRH antagonist protocol was used for ovarian stimulation. Recombinant follicle stimulation hormone (FSH) (Gonafen, Pricon) and/or human menopausal gonadotrophin (Livzon, China) was commenced on day 2 or day 3 of the cycle at a dose of 75–225 IU, and the doses were adjusted according to the ovarian response (follicle count under ultrasound and/or serum E2 levels). When at least two leading follicles reached a size of around 18 mm, ovulation was induced by the administration of recombinant human chorionic gonadotropin (5000–6500 IU, Livzon, China); then, the oocyte was retrieved 36 to 38 h later. Fertilization was conducted by either conventional IVF or by an intracytoplasmic sperm injection (ICSI). Two embryos at the cleavage stage were transferred. The primary outcome was twin pregnancy defined as two live births after 22 weeks of gestation. Secondary outcomes included preterm birth (before 37 completed weeks of gestational age) and low birth weight (birth weight lower than 2500 g). The poor-type endometrium was defined as a multilayered endometrium consisting of prominent outer and midline hyperechogenic lines and inner hypoechogenic regions [37]. Cleavage embryos were graded according to the Istanbul consensus with 1 to 4 grade [38]. Grade I-II embryos were considered as good quality embryos, while Grade III-IV were considered as Poor-quality embryos. Thin endometrium was defined as endometrial thickness lower than 7 mm. Endometrial thickness at 7–14 mm was considered as the moderate endometrium and higher than 14 mm was considered as the thick emdometrium. High education was defined as a master degree or PhD degree. The number of oocyte retrieved at 0–5, 6–15, and > 16 were considered as poor ovarian response, moderate response, and high response, respectively.

In this study, we showed that women with age over 35, one good-quality embryo and none good-quality embryo were associated with reduced rate for twin pregnancy (per cycle and per pregnancy). There was a trend towards an increased rate of twin pregnancy in women with high education and women with GnRH agonist.

Previous studies demonstrated that the good-quality embryos and a high number of retrieved oocytes increase the chance of dizygotic twinning after IVF with DET [32, 39‐42]. Consistent with previous studies, our data demonstrated that women transferred with two good-quality embryos showed higher rates of twin pregnancy compared with women transferred with one or none good-quality embryo. However, our study demonstrated that women with high ovarian response were not associated with increased rates of twin pregnancy. Due to the inadvanced embryo frozen technology in the past decade, the strategy of transferring multiple embryos is often preferred, which leads to increased rates of twin pregnancy. Our research included women who underwent fresh embryo transfer, and if the number of oocytes retrieved was greater than 20, we generally chose to freeze all embryos instead of fresh transfer. Table 1 showed that the proportion of women with high ovarian response is relatively low and the average number of oocytes is around 11. On the other hand, previous studies have often decided to transfer two or more embryos in women with a large number of oocytes, which leads to an increase rate of twin pregnancy [42, 43]. These data may explain the difference between our study and previous studies regarding to the association of high response with twin pregnancy rate. Furthermore, our data also demonstrated that women with good-type endometrium and age under 35 were associated with a higher rate of twin pregnancy, which have not been determined in previous studies. Therefore, these women need to be cautious when choosing double embryo transfer because the rates of twin pregnancy tend to increase in these women.

At the same time, we also explored the risk factors of premature birth and low birth weight infants. Our datas showed that women with age under 35 and women transferred with two good-quality embryos were associated with a higher rate of twin pregnancy as well as preterm birth and low birth weight. In addition, previous studies have shown that twin pregnancy may lead to premature birth and low birth weight infants [44, 45]. Overall, these data strongly suggested the adverse outcomes in women with age under 35 and women transferred with two good-quality embryos. Thus, an elective single embryo transfer should be considered in women with age under 35 and with good-quality embryos. Additionally, there is a trend towards an increased risk of twin pregnancy, perterm birth, and low birth weight in women with high education and women with GnRH agonist use. One study explored the association between educational attainment and twin pregnancies, and the study found no significant association [32]. Our study found there is a trend towards an increased risk of twin pregnancy in women with high education, the result could be random and should be further confirmed by future studies with large sample size. An elective single embryo transfer should also be preferred in these women.

Our study has several limitaions. Duo to the retrospective nature, some potential factors that may be associated with the studied outcomes were not included. Additionally, the obstetric outcomes were not available to analyse in our dataset.

In the present study, we showed that women with age over 35, one good-quality embryo and none good-quality embryo were associated with reduced rate of twin pregnancy (per cycle and per pregnancy). There is a trend towards an increased rate of twin pregnancy in women with high education and women with GnRH agonist.