Diverse impacts of female chromosomal polymorphisms on assisted reproduction outcomes: a retrospective cohort study.

This study is a retrospective cohort study. A total of 11,739 infertile couples who received IVF/ICSI treatment and karyotyping at Peking University Third Hospital between 2015 and 2021 were included. The control group consisted of couples with normal karyotypes and the FCP group included couples in which the female partner carried chromosomal polymorphism(s) and the male partner had a normal karyotype. The following couples were excluded, (i) couples with chromosome aberration, mosaic karyotype, or monogenic disease, (ii) couples in which the male partner carried chromosomal polymorphism(s), (iii) couples received gamete donation. The study incorporated the initial ovarian stimulation cycle and the initial corresponding embryo transfer cycle (fresh or frozen) for each couple. The Peking University Third Hospital Medical Science Research Ethics Committee granted ethical approval for this study (IRB00006761-M2023384). Informed consent exemptions were approved by the ethics committees due to the retrospective nature of this study. G-banding karyotype with a resolution of 400–550 bands was performed according to standard procedures. At least 20 metaphases were examined for each participant. The karyotyping results were reviewed by two experienced cytogeneticists independently. In accordance with the International System for Chromosome Nomenclature 2013 [ 1 ], chromosomal polymorphisms were reported when the heterochromatin exhibited a size greater than twice that of their homologous counterparts. The qh+/− represents the variations in the pericentric heterochromatin of chromosomes 1, 9, and 16. The pstk+/−, pss, and ps + represent the variations of the stalk and satellites on the short arms of the acrocentric chromosomes 13, 14, 15, 21, and 22. The inv(9) is the inversion within the pericentric heterochromatin of chromosome 9. Other inversions within the pericentric heterochromatin of chromosomes 1, 2, 3, 10, and 16 are also regarded as polymorphisms. The cenh+ represents an increase in the size of centromeric heterochromatin. Fig. S 1 displayed representative images of chromosomal polymorphisms. The age of females and males, body mass index (BMI) of females and males, basal endocrine level (follicle stimulating hormone (FSH) and E2), antral follicle count (AFC), type (primary or secondary) and cause (tubal factor, diminished ovarian reserve, polycystic ovary syndrome (PCOS), endometriosis, other maternal factors, and paternal factor) of infertility, and various treatment parameters (methods of stimulation, fertilization, and embryo transfer; stage and number of the transferred embryo) were collected as baseline data. Definitions and assessments of the IVF/ICSI embryological outcomes and clinical outcomes were performed as previously described [ 17 ]. Briefly, 36 hours after the trigger of human chorionic gonadotropin (HCG), the oocytes were retrieved. The presence of a first polar body (PB) indicated mature oocytes (MII oocytes), and the oocyte maturation rate was the proportion of MII oocytes relative to the total number of retrieved oocytes. Normal fertilization was identified by the observation of a second polar body (PB) and two pronuclei (PN) within 16 to 18 hours following insemination. The normal fertilization rate was calculated as the proportion of oocytes exhibiting normal fertilization to the total number of oocytes inseminated. Embryo quality was evaluated 67 to 69 hours post-insemination (Day 3) based on cell count and cytoplasmic fragmentation extent. The transplantable embryo rate was derived by calculating the ratio of embryos that advanced from the 2PN oocytes and attained a stage of five or more cells with cytoplasmic fragmentation not exceeding 30% on Day 3, to the number of embryos that displayed cleavage on Day 2. For the assessment of clinical outcomes, biochemical pregnancy was defined by a serum β-hCG level of more than 10 IU/L, measured 14 days after the transfer of embryos. Clinical pregnancy was confirmed via ultrasound by observing at least one gestational sac 30 days after the embryo transfer. Following the expert consensus and guidelines from China, miscarriage refers to the loss of a pregnancy before 28 weeks of pregnancy [ 18 ]. Preterm birth refers to the parturition before 37 weeks of pregnancy [ 19 ]. A live birth was defined as the successful delivery of one or more live neonates. The denominator for calculating the biochemical pregnancy rate, clinical pregnancy rate, and live birth rate was the number of couples receiving embryo transfer, while the denominators for calculating the miscarriage rate and preterm birth rate were couples with clinical pregnancy and those with successful delivery, respectively. Given that the continuous variables in the present study were non-normally distributed, they were reported as the median with the 25th and 75th percentiles, and their comparisons were conducted using the Mann-Whitney U test or the Kruskal-Wallis test. Categorical variables were presented as the number and percentage, and their comparisons were performed using the Chi-square test or Fisher’s exact test. The significantly different variables ( P  <  0.05) were selected as the confounders for adjustment in the multivariate analyses. Generalized linear regression models with adjustments for potential confounders were employed to compare the embryological outcomes of IVF/ICSI and yielded estimated marginal means (EMMs)(the adjusted means), coefficients, and adjusted P values. Log-binomial regression models with adjustments for potential confounders were applied to evaluate the clinical outcomes of IVF/ICSI, and the adjusted risk ratios (aRRs) and adjusted P values were obtained. Statistical analyses were performed with SPSS version 29.0 (IBM, Inc.). P  < 0.05 was considered statistically significant.

Figure 1 displayed the distribution of couples at various stages of IVF/ICSI treatment. The FCP group included 951 couples and the control group included 10,788 couples (Table 1 ). The two groups were comparable in terms of maternal age, paternal age, maternal BMI, paternal BMI, basal FSH level, basal E2 level, AFC, infertility type, tubal factor, PCOS, endometriosis, other maternal factors, stimulation protocol, fertilization type, embryo transfer method, stage of transferred embryo, and number of transferred embryo (Table 1 ). In the FCP group, the proportions of couples with diminished ovarian reserve (10.7% vs. 13.4%, P  = 0.020, Table 1 ) and paternal infertility factor (47.2% vs. 52.1%, P  = 0.004, Table 1 ) were lower than those in the control group. These two variables would be adjusted in the following multivariate analysis models. According to the types of polymorphisms, the FCP group was divided into five subgroups: qh + (352, 37.0%), pstk+(307, 32.3%), inv(9)(118, 12.4%), multiple (including couples who carried two or more FCPs)(91, 9.6%), and others (including couples who carried other less common FCPs such as ps+, inv(1)(p13q21), etc.)(83, 8.7%) (Table 1 ). Fig. 1 Flow chart of participants at each stage of IVF/ICSI treatment Table 1 Comparison of baseline characteristics between the control and FCP group Control n  = 10,788 FCP group n  = 951 P value Maternal age, years 32.0(30.0 ~ 35.0) 32.0(30.0 ~ 35.0) 0.542 Paternal age, years 33.0(30.0 ~ 36.0) 33.0(30.0 ~ 36.0) 0.390 Maternal BMI, kg/m2 22.2(20.2 ~ 24.8) 22.0(20.2 ~ 25.0) 0.786 Paternal BMI, kg/m2 25.1(23.0 ~ 27.7) 25.1(23.0 ~ 27.8) 0.734 Basal FSH level, IU/L 6.3(4.8 ~ 7.9) 6.2(4.9 ~ 7.9) 0.705 Basal E2 level, pmol/L 155.0(114.0 ~ 200.0) 152.0(114.0 ~ 198.0) 0.671 AFC 11.0(8.0 ~ 15.0) 10.0(8.0 ~ 15.0) 0.752 Infertility type 0.653 Primary 6354(58.9) 553(58.1) Secondary 4434(41.1) 398(41.9) Tubal factor 0.908 No 6770(62.8) 595(62.6) Yes 4018(37.2) 356(37.4) PCOS 0.312 No 8878(82.3) 795(83.6) Yes 1910(17.7) 156(16.4) Diminished ovarian reserve 0.020 No 9345(86.6) 849(89.3) Yes 1443(13.4) 102(10.7) Endometriosis 0.747 No 9686(89.8) 857(90.1) Yes 1102(10.2) 94(9.9) Other maternal factors 0.869 No 8711(80.7) 770(81.0) Yes 2077(19.3) 181(19.0) Paternal factor 0.004 No 5167(47.9) 502(52.8) Yes 5621(52.1) 449(47.2) Stimulation protocol 0.301 GnRH agonist 3689(34.2) 341(35.9) GnRH antagonist 7099(65.8) 610(64.1) Fertilization type 0.690 IVF 7407(68.7) 647(68.0) ICSI 3381(31.3) 304(32.0) Embryo transfer method 0.480 Fresh 6769(62.7) 605(63.6) Frozen 2529(23.4) 228(24.0) No transfer 1490(13.8) 118(12.4) Stage of transferred embryo 0.474 Cleavage stage 8041(86.5) 713(85.6) Blastocyst stage 1257(13.5) 120(14.4) Number of transferred embryo 0.538 one 1991(21.4) 186(22.3) two 7307(78.6) 647(77.7) Type of FCPs qh+ NA 352(37.0) pstk+ NA 307(32.3) inv(9) NA 118(12.4) multiple NA 91(9.6) others NA 83(8.7) FCP female chromosomal polymorphism, BMI body mass index, FSH follicle stimulating hormone, AF , antral follicle count, PCOS polycystic ovarian syndrome, IVF in vitro fertilization, ICSI intracytoplasmic sperm injection, NA not applicable Continuous variables were displayed as the median along with the 25th and 75th percentiles, and their comparisons were conducted using the Mann-Whitney U test. Categorical variables were presented as the number and percentage, and their comparisons were performed using the Chi-square test P values less than 0.050 were shown in bold Flow chart of participants at each stage of IVF/ICSI treatment Comparison of baseline characteristics between the control and FCP group FCP female chromosomal polymorphism, BMI body mass index, FSH follicle stimulating hormone, AF , antral follicle count, PCOS polycystic ovarian syndrome, IVF in vitro fertilization, ICSI intracytoplasmic sperm injection, NA not applicable Continuous variables were displayed as the median along with the 25th and 75th percentiles, and their comparisons were conducted using the Mann-Whitney U test. Categorical variables were presented as the number and percentage, and their comparisons were performed using the Chi-square test P values less than 0.050 were shown in bold Our results showed that there were no significant differences in embryological outcomes (oocytes retrieved, normal fertilization rate, cleavage rate, and transplantable embryo rate) (Table S 1 ) and clinical outcomes (biochemical pregnancy rate, clinical pregnancy rate, miscarriage rate, preterm birth rate, and live birth rate) (Table S 2 ) between the FCP and control group. Regarding the subgroups, none of them exhibited worse IVF/ICSI outcomes (Table S 3 , Table 2 , and Table 3 ). The live birth rate of the qh + subgroup was even higher than that of the control group, after adjustment for AFC, diminished ovarian reserve, and paternal factor (24.7% vs. 19.1%, aRR(95%CI) = 1.28(1.05 ~ 1.56), adjusted P  = 0.014, Table 3 ). Table 2 Comparison of embryological outcomes between the control and FCP subgroups EMM ± SEM Coefficient (95% CI) Adjusted P value Oocytes retrieved Control 12.1 ± 0.1 Ref. Ref. qh+ 11.5 ± 0.4 −0.59(−1.30 ~ 0.12) 0.103 pstk+ 12.4 ± 0.4 0.35(−0.41 ~ 1.11) 0.363 inv(9) 11.9 ± 0.6 −0.19(−1.41 ~ 1.02) 0.754 multiple 13.0 ± 0.7 0.95(− 0.43 ~ 2.33) 0.175 others 11.9 ± 0.7 −0.20(− 1.65 ~ 1.24) 0.783 Normal fertilization rate (%) Control 60.6 ± 0.2 Ref. Ref. qh+ 61.6 ± 1.3 0.01(−0.02 ~ 0.04) 0.466 pstk+ 60.6 ± 1.4 0.00(−0.03 ~ 0.03) 0.974 inv(9) 65.0 ± 2.3 0.04(0.00 ~ 0.09) 0.063 multiple 56.6 ± 2.6 −0.04(−0.09 ~ 0.01) 0.124 others 57.8 ± 2.8 −0.03(− 0.08 ~ 0.03) 0.298 Cleavage rate (%) Control 97.9 ± 0.1 Ref. Ref. qh+ 98.2 ± 0.5 0.00(−0.01 ~ 0.01) 0.487 pstk+ 97.3 ± 0.5 −0.01(− 0.02 ~ 0.00) 0.258 inv(9) 97.5 ± 0.8 0.00(−0.02 ~ 0.01) 0.595 multiple 98.2 ± 0.9 0.00(−0.02 ~ 0.02) 0.773 others 96.8 ± 1.0 −0.01(− 0.03 ~ 0.01) 0.260 Transplantable embryo rate (%) Control 54.3 ± 0.3 Ref. Ref. qh+ 54.2 ± 1.7 0.00(−0.03 ~ 0.03) 0.958 pstk+ 52.0 ± 1.8 −0.02(− 0.06 ~ 0.01) 0.205 inv(9) 58.1 ± 2.8 0.04(−0.02 ~ 0.09) 0.183 multiple 49.4 ± 3.2 −0.05(− 0.11 ~ 0.01) 0.128 others 58.5 ± 3.4 0.04(−0.03 ~ 0.11) 0.222 FCP female chromosomal polymorphism, EMM estimated marginal mean, SEM standard error of the means, CI confidence interval, Ref reference The EMMs, coefficients with their corresponding 95% CIs, and adjusted P values were calculated using generalized linear regression models. The embryological outcomes were adjusted for AFC, diminished ovarian reserve, and paternal factor, except that the oocytes retrieved were adjusted only for AFC and diminished ovarian reserve Table 3 Comparison of clinical outcomes between the control and FCP subgroups a Rate (cases/study subjects (%)) aRR (95% CI) Adjusted P value Biochemical pregnancy Control 3461/9298(37.2) Ref. Ref. qh+ 125/312(40.1) 1.06(0.93 ~ 1.22) 0.389 pstk+ 98/269(36.4) 0.97(0.83 ~ 1.14) 0.707 inv(9) 39/100(39.0) 1.02(0.79 ~ 1.30) 0.903 multiple 34/78(43.6) 1.17(0.91 ~ 1.51) 0.212 others 33/74(44.6) 1.17(0.91 ~ 1.50) 0.219 Clinical pregnancy Control 2861/9298(30.8) Ref. Ref. qh+ 104/312(33.3) 1.07(0.91 ~ 1.26) 0.402 pstk+ 83/269(30.9) 1.00(0.83 ~ 1.19) 0.957 inv(9) 33/100(33.0) 1.04(0.79 ~ 1.38) 0.789 multiple 26/78(33.3) 1.08(0.79 ~ 1.48) 0.614 others 29/74(39.2) 1.26(0.95 ~ 1.67) 0.113 Miscarriage Control 951/2861(33.2) Ref. Ref. qh+ 25/104(24.0) 0.73(0.52 ~ 1.04) 0.079 pstk+ 28/83(33.7) 1.02(0.75 ~ 1.39) 0.890 inv(9) 12/33(36.4) 1.11(0.71 ~ 1.75) 0.650 multiple 8/26(30.8) 0.93(0.52 ~ 1.66) 0.808 others 7/29(24.1) 0.72(0.38 ~ 1.38) 0.325 Preterm birth Control 291/1772(16.4) Ref. Ref. qh+ 12/77(15.6) 0.94(0.55 ~ 1.60) 0.824 pstk+ 10/50(20.0) 1.21(0.69 ~ 2.13) 0.512 inv(9) 1/20(5.0) 0.30(0.04 ~ 2.01) 0.212 multiple 5/16(31.3) 1.85(0.89 ~ 3.88) 0.101 others 2/19(10.5) 0.65(0.18 ~ 2.43) 0.524 Live birth Control 1767/9268(19.1) Ref. Ref. qh+ 77/312(24.7) 1.28(1.05 ~ 1.56) 0.014 pstk+ 49/269(18.2) 0.95(0.73 ~ 1.22) 0.672 inv(9) 20/100(20.0) 1.02(0.69 ~ 1.51) 0.919 multiple 16/77(20.8) 1.10(0.71 ~ 1.70) 0.663 others 19/73(26.0) 1.37(0.93 ~ 2.01) 0.115 a The number of couples at each stage was depicted in Fig. 1 , while the methods employed for calculation were elucidated in the Materials and Methods Section. Couples who were lost to follow-up were excluded from the calculation of the live birth rate FCP female chromosomal polymorphism, aRR adjusted risk ratio, CI confidence interval, Ref reference Log-binomial regression models with adjustment for AFC, diminished ovarian reserve, and paternal factor were employed to calculate the aRRs with their corresponding 95% CIs and adjusted P values P values less than 0.050 were shown in bold Comparison of embryological outcomes between the control and FCP subgroups FCP female chromosomal polymorphism, EMM estimated marginal mean, SEM standard error of the means, CI confidence interval, Ref reference The EMMs, coefficients with their corresponding 95% CIs, and adjusted P values were calculated using generalized linear regression models. The embryological outcomes were adjusted for AFC, diminished ovarian reserve, and paternal factor, except that the oocytes retrieved were adjusted only for AFC and diminished ovarian reserve Comparison of clinical outcomes between the control and FCP subgroups a a The number of couples at each stage was depicted in Fig. 1 , while the methods employed for calculation were elucidated in the Materials and Methods Section. Couples who were lost to follow-up were excluded from the calculation of the live birth rate FCP female chromosomal polymorphism, aRR adjusted risk ratio, CI confidence interval, Ref reference Log-binomial regression models with adjustment for AFC, diminished ovarian reserve, and paternal factor were employed to calculate the aRRs with their corresponding 95% CIs and adjusted P values P values less than 0.050 were shown in bold Generally, couples with poor semen quality or experiencing fertilization failure are more prone to receive ICSI treatment, thereby resulting in significant heterogeneity between couples undergoing IVF treatment and those undergoing ICSI treatment. Indeed, an examination of 10,788 control couples unveiled significant disparities in baseline characteristics, such as age, basal FSH, AFC, infertility type, infertility factors, stimulation protocol, and stage of the transferred embryo, between those undergoing IVF treatment ( n  = 7407) and those undergoing ICSI treatment ( n  = 3381) (Table S 4 ). Therefore, we next investigated the impacts of FCPs on the outcomes of IVF and ICSI, respectively. Fig. S 2 displayed the distribution of couples at various stages of IVF treatment. In couples undergoing IVF, the FCP group ( n  = 647) and the control group (n = 7407) differed significantly in the proportions of couples with diminished ovarian reserve and male infertility factors (Table S 5 ), which would be adjusted in subsequent multivariate analyses. When the FCPs were analyzed as a whole, the embryological and clinical outcomes of the FCP group were not significantly different from the control group, except that the FCP group exhibited a higher live birth rate (22.7% vs. 19.0%, aRR(95%CI) = 1.18(1.01 ~ 1.39), adjusted P  = 0.040) (Table S 6 and Table S 7 ). Further comprehensive examinations on the effects of various subgroups of FCPs revealed significantly increased normal fertilization rate (63.0% vs. 59.2%, coefficient (95%CI) = 0.04 (0.01 ~ 0.07), adjusted P  = 0.022), clinical pregnancy rate (37.0% vs. 30.7%, aRR(95%CI) = 1.20(1.00 ~ 1.44), adjusted P  = 0.048), and live birth rate (27.0% vs.19.0%, aRR(95%CI) = 1.42(1.13 ~ 1.78), adjusted P  = 0.003) in the qh + subgroup compared to the control group (Table S 8 , Table 4 , and Table 5 ). However, the presence of multiple polymorphisms in women (the “multiple” subgroup) increased the risk of preterm birth (62.5% vs. 16.9%, aRR(95%CI) = 3.71(2.14 ~ 6.43), adjusted P  < 0.001, Table 5 ). The IVF outcomes of the pstk+ subgroup, inv(9) subgroup, and ‘others’ subgroup were not significantly different from those of the control group (Table 4 and Table 5 ). These results indicated that various types of FCPs affected IVF outcomes differently. Table 4 Comparison of embryological outcomes between the control and FCP subgroups in couples undergoing IVF EMM ± SEM Coefficient (95% CI) Adjusted P value Oocytes retrieved Control 11.7 ± 0.1 Ref. Ref. qh+ 10.9 ± 0.5 − 0.76(−1.73 ~ 0.22) 0.128 pstk+ 12.2 ± 0.5 0.48(−0.54 ~ 1.50) 0.357 inv(9) 12.2 ± 0.9 0.49(−1.22 ~ 2.19) 0.578 multiple 12.6 ± 1.0 0.90(−0.99 ~ 2.79) 0.352 others 10.8 ± 1.0 −0.88(−2.78 ~ 1.03) 0.368 Normal fertilization rate (%) Control 59.2 ± 0.3 Ref. Ref. qh+ 63.0 ± 1.6 0.04(0.01 ~ 0.07) 0.022 pstk+ 59.6 ± 1.7 0.00(−0.03 ~ 0.04) 0.819 inv(9) 64.2 ± 2.9 0.05(−0.01 ~ 0.11) 0.085 multiple 56.4 ± 3.2 −0.03(− 0.09 ~ 0.04) 0.386 others 57.7 ± 3.2 −0.02(− 0.08 ~ 0.05) 0.639 Cleavage rate (%) Control 97.6 ± 0.1 Ref. Ref. qh+ 98.4 ± 0.6 0.01(0.00 ~ 0.02) 0.202 pstk+ 96.9 ± 0.6 −0.01(−0.02 ~ 0.01) 0.259 inv(9) 96.6 ± 1.1 −0.01(− 0.03 ~ 0.01) 0.358 multiple 97.4 ± 1.2 0.00(−0.03 ~ 0.02) 0.863 others 95.6 ± 1.2 −0.02(− 0.04 ~ 0.00) 0.099 Transplantable embryo rate (%) Control 52.4 ± 0.4 Ref. Ref. qh+ 52.2 ± 1.9 0.00(−0.04 ~ 0.04) 0.922 pstk+ 50.9 ± 2.1 −0.02(− 0.06 ~ 0.03) 0.470 inv(9) 54.7 ± 3.4 0.02(−0.05 ~ 0.09) 0.503 multiple 47.2 ± 3.8 −0.05(− 0.13 ~ 0.02) 0.175 others 53.8 ± 3.8 0.01(−0.06 ~ 0.09) 0.713 FCP female chromosomal polymorphism, EMM estimated marginal mean, SEM standard error of the means, CI confidence interval, Ref reference The EMMs, coefficients with their corresponding 95% CIs, and adjusted P values were calculated using generalized linear regression models. The embryological outcomes, except for the oocytes retrieved, were adjusted for the paternal factor P values less than 0.050 were shown in bold Table 5 Comparison of clinical outcomes between the control and FCP subgroups in couples undergoing IVF a Rate (cases/study subjects (%)) aRR (95% CI) Adjusted P value Biochemical pregnancy Control 2381/6379(37.3) Ref. Ref. qh+ 91/211(43.1) 1.15(0.98 ~ 1.34) 0.088 pstk+ 69/189(36.5) 0.98(0.81 ~ 1.18) 0.802 inv(9) 26/62(41.9) 1.13(0.84 ~ 1.52) 0.412 multiple 22/53(41.5) 1.10(0.80 ~ 1.52) 0.548 others 23/54(42.6) 1.14(0.83 ~ 1.55) 0.426 Clinical pregnancy Control 1960/6379(30.7) Ref. Ref. qh+ 78/211(37.0) 1.20(1.00 ~ 1.44) 0.048 pstk+ 58/189(30.7) 1.00(0.80 ~ 1.24) 0.992 inv(9) 24/62(38.7) 1.27(0.92 ~ 1.74) 0.143 multiple 16/53(30.2) 0.98(0.65 ~ 1.48) 0.914 others 22/54(40.7) 1.32(0.96 ~ 1.83) 0.090 Miscarriage Control 644/1960(32.9) Ref. Ref. qh+ 21/78(26.9) 0.82(0.57 ~ 1.19) 0.298 pstk+ 17/58(29.3) 0.89(0.60 ~ 1.34) 0.586 inv(9) 9/24(37.5) 1.14(0.68 ~ 1.92) 0.626 multiple 7/16(43.8) 1.34(0.76 ~ 2.34) 0.312 others 7/22(31.8) 0.97(0.52 ~ 1.79) 0.916 Preterm birth Control 205/1212(16.9) Ref. Ref. qh+ 9/57(15.8) 0.94(0.51 ~ 1.74) 0.848 pstk+ 6/39(15.4) 0.92(0.44 ~ 1.94) 0.821 inv(9) 1/14(7.1) 0.42(0.06 ~ 2.78) 0.368 multiple 5/8(62.5) 3.71(2.14 ~ 6.43) < 0.001 others 2/12(16.7) 0.98(0.28 ~ 3.49) 0.975 Live birth Control 1209/6362(19.0) Ref. Ref. qh+ 57/211(27.0) 1.42(1.13 ~ 1.78) 0.003 pstk+ 38/189(20.1) 1.06(0.79 ~ 1.41) 0.704 inv(9) 14/62(22.6) 1.19(0.75 ~ 1.90) 0.458 multiple 8/53(15.1) 0.79(0.42 ~ 1.50) 0.467 others 12/53(22.6) 1.19(0.72 ~ 1.96) 0.494 a The number of couples at each stage was depicted in Fig. S 2 , while the methods employed for calculation were elucidated in the Materials and Methods Section. Couples who were lost to follow-up were excluded from the calculation of the live birth rate FCP female chromosomal polymorphism, aRR adjusted risk ratio, CI confidence interval, Ref reference Log-binomial regression models with adjustment for paternal factor were employed to calculate the aRRs with their corresponding 95% CIs and adjusted P values P values less than 0.050 were shown in bold Comparison of embryological outcomes between the control and FCP subgroups in couples undergoing IVF FCP female chromosomal polymorphism, EMM estimated marginal mean, SEM standard error of the means, CI confidence interval, Ref reference The EMMs, coefficients with their corresponding 95% CIs, and adjusted P values were calculated using generalized linear regression models. The embryological outcomes, except for the oocytes retrieved, were adjusted for the paternal factor P values less than 0.050 were shown in bold Comparison of clinical outcomes between the control and FCP subgroups in couples undergoing IVF a a The number of couples at each stage was depicted in Fig. S 2 , while the methods employed for calculation were elucidated in the Materials and Methods Section. Couples who were lost to follow-up were excluded from the calculation of the live birth rate FCP female chromosomal polymorphism, aRR adjusted risk ratio, CI confidence interval, Ref reference Log-binomial regression models with adjustment for paternal factor were employed to calculate the aRRs with their corresponding 95% CIs and adjusted P values P values less than 0.050 were shown in bold Fig. S 3 displayed the distribution of couples at various stages of ICSI treatment. In couples undergoing ICSI, the baseline characteristics were comparable between the FCP group ( n  = 304) and the control group ( n  = 3381) (Table S 9 ). Notably, the FCP group as a whole showed a significantly lower oocyte maturation rate (76.0% vs. 78.8%, coefficient (95%CI) = − 0.03(− 0.05 ~ − 0.01), P  = 0.008, Table S 10 ), while it was not significantly different from the control group in other embryological outcomes and clinical outcomes (Table S 10 and Table S 11 ). Further analyses on different subgroups of FCPs revealed that the oocyte maturation rate in the ‘others’ subgroup was significantly lower than that in the control group (69.0 vs. 78.8%, coefficient (95%CI) = − 0.10(− 0.17 ~ − 0.03), P  = 0.008, Table S 12 and Table 6 ). A similar trend was also observed in the qh + subgroup, pstk+ subgroup, inv(9) subgroup, and ‘multiple’ subgroup, albeit not reaching statistical significance (Table 6 ). These results suggested that FCPs, particularly those less prevalent, exert detrimental effects on oocyte maturation. In addition, the preterm birth rate of the pstk+ subgroup was significantly higher than that of the control group (36.4% vs. 15.4%, aRR = 2.37 (1.06 ~ 5.30), P  = 0.036, Table 7 ). Interestingly, in contrast to the results observed in couples undergoing IVF, the qh + subgroup in couples undergoing ICSI exhibited a lower normal fertilization rate (58.8% vs. 63.8%, coefficient (95%CI) = − 0.05(− 0.10 ~ 0.00), P  = 0.032, Table 6 ), a comparable clinical pregnancy rate (25.7% vs. 30.9%, RR = 0.83(0.60 ~ 1.17), P  = 0.289, Table 7 ), and a comparable live birth rate (19.8% vs. 19.2%, RR = 1.03(0.69 ~ 1.54), P  = 0.880, Table 7 ), compared with the control group (Table 7 ). These results suggested that the same FCP could affect the outcomes of IVF and ICSI very differently and qh + in females compromised the effectiveness of ICSI treatment rather than IVF treatment. Table 6 Comparison of embryological outcomes between the control and FCP subgroups in couples undergoing ICSI EMM ± SEM Coefficient (95% CI) P value Oocytes retrieved Control 12.8 ± 0.1 Ref. Ref. qh+ 12.7 ± 0.7 −0.15(−1.52 ~ 1.21) 0.828 pstk+ 13.2 ± 0.8 0.38(− 1.15 ~ 1.90) 0.629 inv(9) 14.0 ± 1.1 1.11(−1.11 ~ 3.33) 0.325 multiple 13.0 ± 1.4 0.16(−2.49 ~ 2.81) 0.906 others 13.9 ± 1.5 1.07(−1.95 ~ 4.10) 0.487 Metaphase II (M II) oocytes Control 9.9 ± 0.1 Ref. Ref. qh+ 9.5 ± 0.6 −0.43(−1.53 ~ 0.66) 0.436 pstk+ 10.1 ± 0.6 0.19(−1.04 ~ 1.41) 0.767 inv(9) 10.7 ± 0.9 0.79(−0.99 ~ 2.56) 0.385 multiple 9.4 ± 1.1 −0.53(−2.66 ~ 1.59) 0.622 others 10.0 ± 1.2 0.02(−2.40 ~ 2.44) 0.986 Oocyte maturation rate (%) Control 78.8 ± 0.3 Ref. Ref. qh+ 75.8 ± 1.6 −0.03(−0.06 ~ 0.00) 0.073 pstk+ 78.2 ± 1.9 −0.01(− 0.04 ~ 0.03) 0.740 inv(9) 76.5 ± 2.7 −0.02(− 0.08 ~ 0.03) 0.397 multiple 74.6 ± 3.2 −0.04(− 0.11 ~ 0.02) 0.197 others 69.0 ± 3.7 −0.10(− 0.17 ~ − 0.03) 0.008 Normal fertilization rate (%) Control 63.8 ± 0.4 Ref. Ref. qh+ 58.8 ± 2.3 −0.05(−0.10 ~ 0.00) 0.032 pstk+ 62.9 ± 2.6 −0.01(− 0.06 ~ 0.04) 0.722 inv(9) 67.2 ± 3.8 0.03(−0.04 ~ 0.11) 0.367 multiple 56.3 ± 4.5 −0.08(− 0.17 ~ 0.01) 0.099 others 57.8 ± 5.2 −0.06(− 0.16 ~ 0.04) 0.253 Cleavage rate (%) Control 98.5 ± 0.1 Ref. Ref. qh+ 97.8 ± 0.7 −0.01(−0.02 ~ 0.01) 0.341 pstk+ 98.3 ± 0.8 0.00(−0.02 ~ 0.01) 0.791 inv(9) 99.1 ± 1.2 0.01(−0.02 ~ 0.03) 0.631 multiple 99.6 ± 1.4 0.01(−0.02 ~ 0.04) 0.439 others 100.0 ± 1.7 0.02(−0.02 ~ 0.05) 0.359 Transplantable embryo rate (%) Control 58.5 ± 0.6 Ref. Ref. qh+ 58.2 ± 3.0 0.00(−0.06 ~ 0.06) 0.914 pstk+ 54.5 ± 3.4 −0.04(− 0.11 ~ 0.03) 0.248 inv(9) 65.3 ± 5.0 0.07(−0.03 ~ 0.17) 0.174 multiple 53.0 ± 5.9 −0.06(− 0.17 ~ 0.06) 0.344 others 71.2 ± 7.0 0.13(−0.01 ~ 0.26) 0.071 FCP female chromosomal polymorphism, EMM estimated marginal mean, SEM standard error of the means, CI confidence interval, Ref reference The EMMs, coefficients with their corresponding 95% CIs, and P values were calculated using generalized linear regression models P values less than 0.050 were shown in bold Table 7 Comparison of clinical outcomes between the control and FCP subgroups in couples undergoing ICSI a Rate (cases/study subjects (%)) RR (95% CI) P value Biochemical pregnancy Control 1080/2919(37.0) Ref. Ref. qh+ 34/101(33.7) 0.91(0.69 ~ 1.20) 0.505 pstk+ 29/80(36.3) 0.98(0.73 ~ 1.32) 0.892 inv(9) 13/38(34.2) 0.93(0.59 ~ 1.44) 0.729 multiple 12/25(48.0) 1.30(0.86 ~ 1.96) 0.214 others 10/20(50.0) 1.35(0.87 ~ 2.10) 0.181 Clinical pregnancy Control 901/2919(30.9) Ref. Ref. qh+ 26/101(25.7) 0.83(0.60 ~ 1.17) 0.289 pstk+ 25/80(31.3) 1.01(0.73 ~ 1.41) 0.941 inv(9) 9/38(23.7) 0.77(0.43 ~ 1.36) 0.365 multiple 10/25(40.0) 1.30(0.80 ~ 2.10) 0.293 others 7/20(35.0) 1.13(0.62 ~ 2.07) 0.681 Miscarriage Control 307/901(34.1) Ref. Ref. qh+ 4/26(15.4) 0.45(0.18 ~ 1.12) 0.085 pstk+ 11/25(44.0) 1.29(0.82 ~ 2.03) 0.267 inv(9) 3/9(33.3) 0.98(0.39 ~ 2.48) 0.963 multiple 1/10(10.0) 0.29(0.05 ~ 1.89) 0.197 others 0/7(0.0) / 0.999 Preterm birth Control 86/560(15.4) Ref. Ref. qh+ 3/20(15.0) 0.98(0.34 ~ 2.82) 0.965 pstk+ 4/11(36.4) 2.37(1.06 ~ 5.30) 0.036 inv(9) 0/6(0.0) NA NA multiple 0/8(0.0) NA NA others 0/7(0.0) NA NA Live birth Control 558/2906(19.2) Ref. Ref. qh+ 20/101(19.8) 1.03(0.69 ~ 1.54) 0.880 pstk+ 11/80(13.8) 0.72(0.41 ~ 1.25) 0.237 inv(9) 6/38(15.8) 0.82(0.39 ~ 1.72) 0.603 multiple 8/24(33.3) 1.74(0.98 ~ 3.07) 0.058 others 7/20(35.0) 1.82(1.00 ~ 3.33) 0.051 a The number of couples at each stage was depicted in Fig. S 3 , while the methods employed for calculation were elucidated in the Materials and Methods Section. Couples who were lost to follow-up were excluded from the calculation of the live birth rate FCP female chromosomal polymorphism, aRR adjusted risk ratio, CI confidence interval, Ref reference, NA not applicable Log-binomial regression models were employed to calculate the aRRs with their corresponding 95% CIs and P values P values less than 0.050 were shown in bold Comparison of embryological outcomes between the control and FCP subgroups in couples undergoing ICSI FCP female chromosomal polymorphism, EMM estimated marginal mean, SEM standard error of the means, CI confidence interval, Ref reference The EMMs, coefficients with their corresponding 95% CIs, and P values were calculated using generalized linear regression models P values less than 0.050 were shown in bold Comparison of clinical outcomes between the control and FCP subgroups in couples undergoing ICSI a a The number of couples at each stage was depicted in Fig. S 3 , while the methods employed for calculation were elucidated in the Materials and Methods Section. Couples who were lost to follow-up were excluded from the calculation of the live birth rate FCP female chromosomal polymorphism, aRR adjusted risk ratio, CI confidence interval, Ref reference, NA not applicable Log-binomial regression models were employed to calculate the aRRs with their corresponding 95% CIs and P values P values less than 0.050 were shown in bold

Chromosomal polymorphisms refer to the variations in the size or structure of heterochromatin regions. The common chromosomal polymorphisms include increased lengths of pericentric heterochromatin (qh+) on chromosomes 1, 9, and 16, increased stalks (pstk+) on chromosomes 13, 14, 15, 21, and 22, and inversion within the pericentric heterochromatin of chromosome 9 (inv(9)(p12q13), abbreviated as inv(9) in this article). Other less common polymorphisms include double or increased satellites (pss or ps+) on chromosomes 13, 14, 15, 21, and 22, inversion within pericentric heterochromatin of chromosomes 1, 2, 3, 10, and 16, etc. [ 1 ]. The females do not involve Y chromosome polymorphisms. Despite being generally regarded as harmless, chromosomal polymorphisms were more readily detected in individuals suffering from reproductive disorders and were linked with adverse outcomes of assisted reproduction [ 2 – 8 ]. In addition, male chromosomal polymorphisms (MCPs) and female chromosomal polymorphisms (FCPs) were reported to exert distinct impacts on the outcomes of reproductive health [ 9 – 12 ], necessitating separate investigations into MCPs and FCPs. This research was dedicated to examining the impacts of FCPs. Compared with couples with normal karyotypes, couples with FCPs exhibited lower fertilization rates (24 couples with female D/G group polymorphisms or 37 couples with female inv(9) vs. 1088 normal couples in [ 12 ]), lower cleavage rates (99 couples with FCPs vs. 400 normal couples in [ 10 ], hereafter abbreviated as 99 vs. 400), lower embryo quality (86 vs. 214 in [ 13 ]), higher miscarriage rates (101 vs. 2704, and 81 vs. 2135 in [ 2 ]), and higher preterm birth rates (101 vs. 2704, 81 vs. 2135, 163 vs. 2188, 45 vs. 921, and 33 vs. 997 in [ 2 ]). However, the results from other studies refuted these findings (82 vs. 1402 in [ 14 ]; 150 vs. 448 in [ 15 ]; 262 vs. 9713, and 311 vs. 10,858 in [ 16 ]). These conflicting conclusions, which derived from studies where the number of couples with FCPs was small and the FCPs were usually analyzed as a whole, are anxiety-provoking for patients and confusing for clinicians. Here, we comprehensively investigated the impacts of various FCPs on IVF/ICSI outcomes in up to 951 couples with FCPs and 10,788 control couples with normal karyotypes and revealed the exact associations of various types of FCPs with different assisted reproduction outcomes.

Our study investigated the influence of FCPs on assisted reproductive outcomes in detail. No significant differences were observed when examining the impact of the overall FCPs on the whole assisted reproduction population. This finding may explain the absence of a relationship between FCPs and assisted reproduction in some previous studies. However, when investigating the effects of various types of FCPs in couples receiving IVF and ICSI treatment respectively, some intriguing results were obtained. A remarkable finding of the present study is that FCPs impair the maturation of oocytes. Constitutive heterochromatin is known to participate in the silencing of gene expression, maintenance of genome stability, and correct chromosome segregation [ 20 – 22 ]. Polymorphisms or anomalies in these regions were reported to be related to chromosome segregation errors and chromosome aneuploidies [ 23 , 24 ]. A previous study showed that women with chromosomal polymorphisms have an increased number of aneuploid blastocysts [ 6 ]. Another study suggested a correlation between FCPs and the occurrence of multinucleated embryos [ 25 ]. Taken together, these findings suggest the hypothesis that FCPs may increase the probability of meiotic errors, leading to a higher prevalence of chromosomal aneuploidy in oocytes and impairing the maturation of oocytes and the subsequent development of embryos. However, this intriguing hypothesis requires further experimental verification. Our findings suggested an increased vulnerability to preterm birth in women undergoing IVF with multiple polymorphisms and in women undergoing ICSI with pstk+. Another study, which did not distinguish between different types of polymorphisms, also indicated a link between FCPs and a higher preterm birth rate [ 2 ]. How FCPs contribute to the increased risk of preterm birth remains uncertain, and whether it is related to the detrimental effects of FCPs on oocyte development, warrants further research. An unexpected finding was the significantly distinct effects of qh + on the outcomes of IVF and ICSI. Our findings demonstrated that women who carried qh + and underwent IVF treatment exhibited higher rates of fertilization, clinical pregnancy, and live birth. A previous study indicated that women with chromosomal polymorphisms had a higher clinical pregnancy rate and live birth rate [ 16 ]. However, this study did not specifically examine the influence of different types of polymorphisms, nor did it separately analyze the impact of polymorphisms on the outcomes of IVF and ICSI. In contrast to the observations in couples undergoing IVF, the present study showed that qh + in females had a detrimental effect on ICSI outcomes, notably reducing the fertilization rate. Hence, for couples who are recommended for ICSI treatment due to poor semen quality in males or previous fertilization failures, the expected effects may not be achieved if the female partners carry qh + . Intriguingly, our recent publication [ 26 ] on MCPs showed that couples who underwent ICSI and carried pstk+ in males also exhibited a higher risk of preterm birth, suggesting that MCPs and FCPs share similar influences in the risk of preterm birth. However, the rare polymorphisms have a significant impact on gamete quality in females, while in males, it is inv(9) and 9qh + that significantly affect gamete quality. In addition, male Yqh + is related to an increased risk of preterm birth in couples undergoing ICSI, suggesting that Y chromosome polymorphisms in males bring additional risks for couples undergoing ICSI. One of the strengths of the present study is that we excluded MCPs and focused on the impact of FCPs. Additionally, the large sample size enables us to explore the associations of different types of FCPs with the outcomes of IVF and ICSI, respectively. However, several limitations should be noted. First, despite our efforts to analyze as many baseline characteristics as possible and adjust for potential confounders, the results may be affected by unidentified confounders. Second, our investigation is a single-center study conducted in the northern Chinese population, limiting the generalizability of the findings to other populations. Third, retrospective study may be subject to recall bias and reverse causation bias, and cannot elucidate the underlying biological mechanisms of the findings. Additionally, the relatively small sample sizes in some subgroups reduce the power of statistical analysis. Therefore, further investigation through larger-scale prospective cohort studies or even randomized controlled trials, coupled with in-depth mechanistic studies, will be beneficial in elucidating the impact of FCPs on assisted reproductive outcomes.

Our study revealed remarkable heterogeneity in the impacts of various FCPs on IVF and ICSI outcomes, and these findings will contribute to developing personalized treatment strategies according to the type of FCPs and the fertilization method. In the future, larger prospective cohort studies, randomized controlled trials, and functional studies are warranted to comprehensively elucidate the impact of each type of FCPs on assisted reproductive health.

Supplementary Material 1. Supplementary Material 1.