Association of HY-restricting HLA Class II Alleles, Sex of Firstborn Child, and Pregnancy Outcome in RPL Patients

In a Danish cohort (Kolte et al., 2016, Nielsen et al., 2009) the first pregnancy outcome after admission and the long term live birth rate were associated with carriage of known HY-r HLA class II alleles in the sRPL patient with a first born boy, but not in the sRPL with a first born girl. The live birth rate in the first pregnancy after admission and the hazard ratio for live birth on longterm were comparable between sRPL with a first born girl and boy if the sRPL did not carry a HY-r HLA class II allele. In contrast, sRPL after a first born boy had a significant poorer prognosis than sRPL after a first born girl when the patients carried ≥1 HY-r HLA class II allele.

These findings were the first of its kind and it has not been confirmed in other cohorts ever since. In a new Danish cohort from another region of Denmark, this study will examine if these findings can be replicated.

Before data collection, a sample size calculation was performed based on the findings in the former studies (Nielsen et al., 2009; Kolte et al., 2016). These studies differ from the present study, since Nielsen et al. (2009) included HLA DRB3*0301 but not HLA DRB1*07 as a HY-r allele, and Kolte et al.(2016) included HLA DRB3*0301 too and measured the cumulative live birth rate in contrast to first pregnancy outcome measured in the present study. However, the sample size calculation based on weighted calculations on results from these studies is the closest we get a suitable sample size for the present study. The sample size in the present study should consist of at least 88 sRPL patients with a first born boy with maternal HY-r HLA class II allele and 73 sRPL patients with a firstborn boy and no maternal HY-r HLA class II alleles on weighted calculations with an alpha level = 0.05, a power of 80 %, and a inclusion ratio of 1.2 more with than without such HLA carriage. The sample size needed to find difference between women with a firstborn boy vs girl with such HLA carriage was smaller; ie. 48 patients for comparing patients with ≥1 HY-r HLA alleles. As this was the primary aim, this sample size was considered sufficient for the study.

Study inclusion started January 1, 2016, and will end when this sample size is reached.

The anticipated sample size of the pRPL group is 1.2 times the total number of sRPL patients since pRPL normally account for about 55 % of RPL patients. Thus, 180 patients are expected in the pRPL, although this is not taking into account when deciding the timing for final follow-up and data collection.

All RPL patients with ≥3 consecutive pregnancy losses admitted to The Center for Recurrent Pregnancy Loss of Western Denmark who do not have significant uterine malformations, prior birth of children of both sexes, or known chromosomal abnormalities will be included. An obstetric and gynecologic history and a routine blood sample will be obtained on all patients at their first visit according to the ESHRE RPL guideline (2018), on which basis the treatment plan will be decided. The majority of patients have a chromosomal analysis on the patient and her partner and all have a 3D ultrasound, HSU or HSG performed.

Both chemical and clinical pregnancies documented in hospital's or practitioner's records are included, while confirmed molar and ectopic pregnancies are not accounted for.

sRPL is defined as ≥3 pregnancy losses after a pregnancy beyond 22 weeks of gestation while pRPL is defined as ≥3 pregnancy losses with no prior pregnancy beyond 22 weeks of gestation.

Only sRPL with birth of children with same sex prior to RPL will be included in the comparison of sRPL patients with previous boy(s) with sRPL patients with previous girl(s) only.

The primary outcome is the association of reproductive outcome in first pregnancy after admission to our RPL Center with the carriage of HY-restricting HLA class II alleles in the following three groups: sRPL having a first born boy and sRPL having a a girl and also in patients with pRPL.

In addition, we will also explore the prevalence of these HY-restricted HLA class II alleles separately and the sex ratio of births after admission in the three groups.

HLA-DRB1 typing is performed based on genetic analyses of peripheral blood as part of the routine work-up at our clinic.

HY restricted HLA class II alleles in our study is defined as DRB1*01/10 (in strong positive linkage disequilibrium with HLA-DQB1*0501 ); HLA-DRB1*15 and HLA-DRB1*07, which to date have been reported to restrict presentation of HY-antigens.

All data is collected in the RPL clinical database of The RPL Center at Aalborg University Hospital, Denmark (Approval number: 2018-5).

Inclusion criteria

• ≥ 3 consecutive pregnancy losses before admission

Exclusion criteria

• Significant uterine malformation
• Chromosomal abnormalities