IVF and ICSI outcomes of female patients with X chromosome mosaicism: A 5-year retrospective cohort study


 BackgroundThis study focused on the assisted reproductive treatment (ART) outcomes of female patients with X chromosome mosaicism (XM), who underwent their first IVF/ICSI and day 2 or day3 fresh embryo transfer, and the possible impacts of the different mosaic types.Results78 couples with XM female and normal male were included as the X group. 78 couples with normal karyotype were included as the control group. Subgroup X1 included 41 45,X/46,XX cases, Subgroup X2 included 23 47,XXX/46,XX cases, and Subgroup X3 included 13 45,X/47,XXX/46,XX cases. With similar female age and similar body mass index (BMI), the X group had higher total gonadotropin (Gn) dosage than the control group (1800 IU VS 1612 IU). In subgroup analysis, the follical number during oocyte retrieval was less in subgroup X1 than that in X2 or X3. The fertilization rate was lower in subgroup X1 than that in subgroup X2. The utilization rate was higher in subgroup X2 than that in subgroup X3. The implantation rate, clinical pregnancy rate, and miscarriage rate before 12 weeks' gestation were similar in all groups.ConclusionsFemale with 45,X cell line may face higher Gn dosage, less follical number during oocyte retrieval and fewer embryos. But female with X chromosome mosaicism may have similar clinical pregnancy rate and miscarriage rate after fresh embryo transfer.


Introduction
Karyotyping is widely used in the diagnosis of chromosome diseases. Aneuploidy, structure variation and mosaicism can be detected [1,2]. Double X chromosomes are essential for female [3][4][5]. Karyotype of infertile female patient could be 46,XX , 45,X , 47,XXX or mosaic type of these [6]. Some cases need to undertake IVF or ICSI to get pregnancy [7]. Whether X chromosome mosaicism (XM) affect the IVF/ICSI outcome was unclear [7,8]. This study included 78 XM cases. Their history and embryo results of the first IVF or ICSI cycle were collected. The pregnancy outcomes were followed till 12 weeks pregnancy.

Karyotype details
The X group was further divided into subgroups ( In the X group, the percentage of 46,XX cells was 78.79%-98.00%. In Subgroup X1, the percentage of 45,X cells among total cells was 3.03%-17.17%. In Subgroup X2, the percentage of 47,XXX cells among total cells was 2.00%-10.00%. In Subgroup X3, the percentage of 45,X cells among total cells was 2.00%-12.00%, and the percentage of 47,XXX cells among total cells was 2.00%-10.00%.

Baseline comparison and stimulation data
The X group and the control group had similar female age, primary/secondary infertility ratio, median of infertility duration, insemination method, height, BMI, basic FSH, ovarian stimulation protocol, times of gestation and times of pregnancy loss before IVF/ICSI treatment (table 2). Although Gn days and E 2 level on trigger day were similar, follical number during oocyte retrieval were also similar, Gn dosage was higher in X group than that in control group (table 4).
The basic information was similar between subgroup X1, X2 and X3 (table 3).

Embryological indexes
The X group and the control group had similar outcome during fertilization and embryo development (table 4).
In subgroup analysis, the follical number during oocyte retrieval was significant less in subgroup X1 than that in X2 or X3. The average fertilized oocyte number was significant less in subgroup X1 than that in X3. The fertilization rate was much lower in subgroup X1 than that in subgroup X2. The average cleavage number was much lower in subgroup X1 than that in subgroup X3. The utilization rate was much higher in subgroup X2 than that in subgroup X3 (table 5).

Pregnancy outcomes
Fresh embryo transfer rate, average ET number, implantation rate, clinical pregnancy rate, miscarriage rate before 12 weeks' gestation were all similar between X group and control group, and also similar between subgroup X1, X2 and X3(table 4,5).

Possible origin of X chromosome mosaicism
Normal female has two copies of X chromosome in all cells. One X chromosome is from the oocyte and the other is from the sperm. XM could be caused by meiosis and/or mitosis error during gamete genesis or in cleavage/ blastula stage [9,10].The individual usually become 45,X/47,XXX or 45,X/47,XXX/46,XX. If one X chromosome was missed in mitosis, 45,X/46,XX or 47,XXX/46,XX (partial trisomy rescue)could be detected.
Also, previous study indicated that low-grade mosaicism for X aneuploidy was an age-related phenomenon not related to reproductive issues such as recurrent abortions [11]. But these cases usually have one X chromosome loss and become 45,X/46,XX.
One observational study indicated that the time of blood sampling in relation to the menstrual cycle can influence lymphocyte X chromosome mosaicism. In the case group (containing 28 women with X mosaicism and recurrent pregnancy loss), the mean value of aneuploid cells in the follicular and luteal phase samples was 10.0 and 6.3 % respectively and in the control group, it was 2.8 and 1.0 % (P < 0.0001). Estrogens are known to selectively influence cell proliferation. Physiological variations of blood hormone concentration might play a role in regulating the level of X chromosome aneuploidy [12].
In this study, we found 4 types of mosaicism, and the percentage of 46,XX cells in peripheral blood lymphocytes was no less than 78%. The menstrual cycle condition of blood sampling was not recorded. Anti-Mullerian hormone (AMH) was not tested. The percentage of every cell line in various tissues were difficult to reveal. The mosaicism was not confirmed by fluorescent in situ hybridization (FISH) or comparative genome hybridization (CMA) of blood lymphocytes or sample from other tissue.

Clinical history of X chromosome mosaicism
A total or partial absence of one X chromosome will affect female health in the whole life [13]. It affects multiple organ systems and the severity of clinical manifestations varies [14][15][16][17][18]. Some manifestations could be adjusted by medical treatment [16,17]. Some female without symptoms or with mild abnormalities could get married and become infertile.
In this study, all patients in X group had spontaneous and complete puberty. The height ranged from 150 to 172 cm. The age ranged from 27 to 48 years old. One patient had cardiac malformations (interventricular septal defect) and had the operation in her childhood. One patient had uterus duplex and cervix duplex. Bone mineral density was not tested. Spontaneous gestation and miscarriage history seemed similar with control group.
During ovarian stimulation, more Gn were needed for X group. The follical number during oocyte retrieval was significant less in subgroup X1 than that in X2 or X3. The average fertilized oocyte number was significant less in subgroup X1 than that in X3. These results indicated that XM, especially 45,X cell line may affect follicular development and oocyte genesis.
The fertilization rate was lower in subgroup X1 than that in subgroup X2. The mechanism was unclear.
Pregnancy outcome after fresh embryo transfer seemed similar in X group and control group. In X group, one patient had preeclampsia during pregnancy, one patient had premature delivery. No congenital abnormality was found in the resulting babies. We failed to collect the karyotype information of the babies or the fetal tissue from spontaneous miscarriage. The cumulative live birth rate was not analyzed.

Genetic consult
Infertile female with mosaic X chromosome was not rare. The chromosome abnormality may affect the health and outcome of assistant reproductive treatment. Genetic consult should be offered to the couple.
The specific comorbidities for women with Turner syndrome such as infertility, cardiac malformations, bone dysgenesis, and autoimmune diseases may depend on a complex relationship between genes as well as transcriptional and epigenetic factors affecting gene expression across the genome [19]. Risks during pregnancy include aortic disorders, hepatic disease, thyroid disease, type 2 diabetes, and cesarean section delivery [3]. Mosaic X chromosome may have various effects according to different karyotype. The risk of premature ovarian failure and cardiac disorders should be mentioned.

Conclusion
Female with mosaic X chromosome (XM) may need more Gn to get similar amount of oocyte. 45,X/46,XX female may face less follical number during oocyte retrieval and fewer embryos. But XM female may have similar clinical pregnancy rate and miscarriage rate after fresh embryo transfer. XM infertile patients should get genetic consult before assistant reproductive treatment. Healthy condition of the mother and baby should be followed.

Study design and experimental subjects
This cohort study was conducted in infertile couples, who underwent their first IVF or ICSI cycle from June 1, 2014, to September 9, 2019. Every couple was assigned a unique sequence number when they decided to undergo IVF/ICSI treatment.

Karyotyping
The protocol for peripheral blood lymphocytes karyotyping has been described elsewhere [9]. Karyotype and chromosome polymorphisms were reported according to the International System for Human Cytogenomic Nomenclature (ISCN). For each patient, no less than 50 metaphase cells were analyzed, and these cells must come from two culture bottles. The exact cell numbers of every kind of karyotype were reported.

Ovarian stimulation and IVF/ICSI-ET
The ovarian stimulation protocol has been described elsewhere [10]. The initial dosage of gonadotrophin depended on the female partner's age, basal follicle stimulating hormone (FSH), antral follicle count, and BMI. The development of follicles was monitored by ultrasound examination and serum E 2 concentration.
The standard operation procedure and indications of oocyte retrieval, insemination method, embryo evaluation, fresh embryo transfer have been described elsewhere [9]. Pregnant patients visited the clinic every two weeks till 12 weeks' gestation, while others were contacted by phone call with nurses. There was no loss of follow-up case in this study.

Indexes of Outcomes
To explore whether XM affects the IVF and ICSI outcome, indexes were analyzed, such as Gn days, Gn dosage, E 2 on trigger day, follical number during oocyte retrieval, average oocyte number, normal fertilization rates, cleavage rates, embryo utilization rates, fresh embryo transfer rates, clinical pregnancy rates (CPR), implantation rates, miscarriage rate before 12 weeks' gestation.

Statistical analysis
Statistical analyses were performed with SPSS 22 software (IBM, Armonk, NY, USA). A difference with a two-sided P value less than 0.05 was considered statistically significant. Data are expressed as mean ± S.E.M. Missing values were ignored directly by default. Where appropriate, Student's t-test or the non-parametric test (the Mann-Whitney U test) was used to analyze numerical data, while Pearson's chi-squared test or Fisher's exact test was used to analyze categorical data. The Kruskal-Wallis one-way analysis of variance by ranks Pairwise Comparisons was used to analyze multiple sets of data. Bonferroni correction was used when needed.